This project involves in vitro and in vivo analyses of the molecular mechanisms and physiological significance of the methylation of eukaryotic DNA using Novikoff rat hepatoma cell cultures and Xenopus laevis whole animal, cell culture, and recombinant DNA systems. We intend to purify to homogeneity DNA methylase(s) from these systems and compare the physicochemical and immunochemical properties of putatively different DNA methylases noted during the cell cycle and in different cellular compartments. The purified enzyme(s) will be used to analyze the molecular mechanisms underlying the observed sequence specificity of DNA methylation and to investigate analogues of S-adenosylhomocysteine as specific inhibitors of the DNA methylation reaction. Purified DNA methylases will also be used in in vitro studies along with artificially (Novikoff cells) or naturally (Xenopus laevis systems) produced homologous non-methylated DNA to determine directly if DNA methylation plays a role in DNA replication, in the transcription of RNA from DNA, or in a eukaryotic DNA modification-restriction process. In vivo studies will include analysis of the effects of non-methylation of DNA upon subsequent DNA replication.